PROJECT SUMMARY Vascular dysfunction represents a critical link between obesity and cardiovascular disease (CVD). The two most clinically relevant manifestations of vascular dysfunction are endothelial dysfunction and arterial stiffness. Despite focused efforts, the underlying cause of these vascular impairments in obese individuals is unclear. The gut microbiota has emerged as an important regulator of human physiology. Deleterious changes to the number or composition of the microbiota, broadly termed dysbiosis, have been implicated in the development of CVD, and more recently, to endothelial dysfunction. However, four fundamental questions regarding the link between dysbiosis and vascular dysfunction remain unstudied: 1) what specific bacterial alterations initiate the development of vascular dysfunction? 2) what circulating factor(s), downstream of dysbiosis, mediates intestinal- vascular crosstalk and hastens the development of vascular dysfunction? 3) in addition to endothelial dysfunction, does dysbiosis also mediate arterial stiffness? 4) are the existing animal data relevant to human vascular dysfunction? The overall goal of the current proposal is to address these four questions and comprehensively examine the link between the gut microbiota and vascular dysfunction. Building upon our recent data, we hypothesize a sequence of events whereby diet-induced obesity causes gut dysbiosis, characterized by reductions in Bifidobacterium, that lead to compromised intestinal barrier function and elevations in circulating lipopolysaccharide (LPS). Once in circulation, LPS activates toll-like receptor 4 (TLR4) on the vasculature, and elicits an inflammatory signaling cascade that ultimately lead to arterial stiffness and endothelial dysfunction. The translational studies proposed herein will test this hypothesis using three innovative, complementary approaches: 1) microbiota transplants 2) selective microbiota enrichment; and 3) colonization of germ free mice with human microbiota samples. In aim 1, microbiota transplants will be performed in control mice (C57); mice with impaired LPS signaling (TLR4-/-); and mice with enhanced LPS signaling (acyloxyacyl hydrolase knockout mice, AOAH-/-) fed either a standard (SD) or western (WD) diet. In aim 2, specific Bifidobacterium populations that are reduced by WD and correlate with vascular dysfunction will be isolated from the microbiota of healthy animals, cultured, then administered to WD-fed mice to examine their vascular protective effects. In aim 3, relations between the gut microbiota and vascular function will be determined in lean and obese individuals; and microbiota samples from these individuals will be used to colonize the intestines of germ free mice. Collectively, these studies will help identify a novel cause of, and therapeutic target for, obesity-related vascular dysfunction with broad and immediate clinical potential.